Continuous flow microfluidic systems and methods for producing self-assembled material particles

Abstract

A continuous flow microfluidic system for continuous flow operation of a microfluidic chip, characterized in that the pulsation rate of the continuous flow formed by the system is 5% or less. [Effects] Even when a fluid sample is introduced into a microfluidic chip at high pressure, self-assembled material particles such as lipid nanoparticles with high size uniformity can be produced well and stably, enabling the mass production of such self-assembled material particles.

Description

Technical Field

[0001] The present invention relates to a microfluidic system suitable for continuous flow operation of a microfluidic chip and a method for producing self-assembled material particles using the microfluidic system.

Background Art

[0002] Microfluidic technology is a technology that can handle various chemical and biological operations such as mixing, reaction, separation, purification, culture, measurement, and detection with extremely small amounts of samples. Microfluidic technology can be utilized in various applications by providing functional regions with various functions, such as reaction regions where reagents are arranged, in a microfluidic chip equipped with a flow path called a microchannel. Examples of the utilization of microfluidic technology include biological substance analysis, DNA testing, drug discovery and pharmaceutical development, environmental analysis, food quality analysis, and measuring instruments.

[0003] In recent years, microfluidic technology has been rapidly spreading in chemical syntheses such as microparticle production and organic synthesis. In particular, self-assembled material particles such as lipid nanoparticles have been increasingly put into practical use as nucleic acid delivery carrier particles in recent years. For example, lipid nanoparticles are used as mRNA delivery carriers in the mRNA vaccine for the novel coronavirus (SARS-CoV-2) that was granted emergency use authorization in 2020. Against this background, attention has been focused on methods for mass-producing self-assembled material particles such as lipid nanoparticles.

[0004] Conventionally, extrusion methods and ultrasonic treatment are known as methods for producing self-assembled material particles such as lipid nanoparticles, but it is difficult to mass-produce particles with high size uniformity by these methods.

[0005] Furthermore, a method for producing lipid nanoparticles using microfluidic technology is also known, and this method makes it possible to produce particles with high size uniformity. For example, Japanese Patent Publication No. 2018-515324 (Patent Document 1) discloses a method for mass production of lipid nanoparticles by parallelizing Dean vortex branching mixers in a system for continuous flow operation of a microfluidic chip. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] Special Publication No. 2018-515324 [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] In the production of lipid nanoparticles using microfluidic technology, it is desirable to continuously feed the fluid sample at a high flow rate to improve productivity. However, when the sample is supplied at high pressure to supply it to the microfluidic chip at a high flow rate, in systems such as the one described in Patent Document 1, the sample is divided by a manifold and supplied to the parallel-arranged microfluidic chips, resulting in a large pressure loss. Therefore, it is not possible to sufficiently reduce fluid pulsation, making it difficult to mass-produce lipid nanoparticles with high size uniformity.

[0008] Furthermore, when a fluid sample is supplied to a microfluidic chip at high pressure, there is a concern that the microfluidic chip may be damaged. The main cause of microfluidic chip damage is that the high-pressure fluid imparts tensile and compressive stress to the microfluidic chip, causing it to deform. Patent Document 1 maintains production volume while reducing the pressure applied to each microfluidic chip by parallelizing the microfluidic chips. However, since the outlets of the parallelized microfluidic chips are unified by a manifold, it is difficult to guarantee that the lipid nanoparticles produced by each microfluidic chip have equivalent properties.

[0009] The present invention has been made in view of the above circumstances, and aims to provide a continuous flow microfluidic system capable of mass-producing self-assembled material particles such as lipid nanoparticles with high size uniformity. [Means for solving the problem]

[0010] The present inventors, having conducted diligent studies to achieve the above objective, have found that in a continuous flow microfluidic system comprising a microfluidic chip having a first supply channel through which a first fluid flows, a second supply channel through which a second fluid flows, and a mixing and diluting channel provided downstream of the confluence of the first and second supply channels for flowing a mixed fluid of the first and second fluids, the first and second fluids are supplied to the first and second supply channels, respectively, by a continuous flow pump and mixed, and the mixed fluid is recovered through a discharge channel connected downstream of the mixing and diluting channel, the present inventors have found that by controlling the pulsation rate of the continuous flow formed by the system to a predetermined value or less, even when a fluid sample is supplied to the microfluidic chip at high pressure, self-assembled materials such as lipid nanoparticles can be produced well and stably using this system, thus completing the present invention.

[0011] Accordingly, the present invention provides the following continuous flow microfluidic systems and methods for producing self-assembled material particles. 1. A continuous flow microfluidic system for continuous flow operation of a microfluidic chip, comprising the following (1) to (4), characterized in that the pulsation rate of the continuous flow formed by the system is 5% or less. (1) A first continuous flow pump that continuously supplies a first fluid from a first reservoir. (2) A second continuous flow pump that continuously supplies the second fluid from the second reservoir. (3) A microfluidic device comprising a microfluidic chip having the following channels (a) to (c). (i) A first supply channel through which the first fluid flows. (b) A second supply channel through which the second fluid flows. (h) A mixing and diluting channel provided downstream of the confluence point where the first supply channel and the second supply channel merge over a certain length, through which a mixed fluid obtained by mixing the first fluid and the second fluid flows. (4) A discharge channel through which the mixed fluid discharged from the microfluidic device flows. 2. A continuous flow microfluidic system comprising a dilution section including a third continuous flow pump that continuously supplies a dilution solution from a third reservoir, and a dilution channel that supplies the dilution solution discharged from the third continuous flow pump downstream of the microfluidic device in the system to mix the dilution solution with the mixed fluid. 3. A system of one or two continuous flow microfluidic devices comprising a waste recovery channel branched from the discharge channel via a waste valve. 4. A continuous flow microfluidic system having a processing capacity of at least 3 L / h per mixing and dilution channel, as specified in 1 to 3 above. 5. A continuous flow microfluidic system according to any one of 1 to 4, wherein at least a portion of the mixing and diluting channel has a bent channel section formed by a plurality of structural elements installed in the channel, and the axial direction of the mixing and diluting channel is the X direction, the width direction of the mixing and diluting channel perpendicular to the X direction is the Y direction, and the width of the mixing and diluting channel upstream of the bent channel section in the Y direction is [y0], the structural elements are arranged to protrude inward from the side walls facing each other along the Y direction in the mixing and diluting channel, and are installed alternately at predetermined intervals along the X direction, and the protrusion width of the structural elements in the Y direction is 1 / 2[y0] or more and less than 1[y0]. 6. A continuous flow microfluidic system according to any of 1 to 5, wherein the microfluidic chip is made of synthetic quartz glass. 7. The microfluidic device comprises a chip holder and a connector, The chip holder comprises a cover and a base that contact the surface of the microfluidic chip, and a fastener that connects and secures the cover and the base, the microfluidic chip is placed between the cover and the base and the cover and base are connected and secured by the fastener, and the microfluidic chip is fixed in close contact with the cover and the base, and the connector penetrates at least one of the cover and the base, with one end connected to an opening provided at the upstream end of the first supply channel, the upstream end of the second supply channel, and the downstream end of the mixing and diluting channel of the microfluidic chip, and the other end forming a supply port for the first fluid or the second fluid or an outlet for the mixed fluid, in a microfluidic device, A continuous flow microfluidic system according to any of 1 to 6, wherein the flatness of the surface of the microfluidic chip in contact with the cover and the flatness of the surface of the microfluidic chip in contact with the base are both 50 μm or less, and the flatness of the surface of the cover in contact with the microfluidic chip and the flatness of the surface of the base in contact with the microfluidic chip are both 50 μm or less. 8. A continuous flow microfluidic system for producing self-assembled material particles, wherein in the mixing and diluting channel, a self-assembled material-containing solution supplied from one of the first supply channel and the second supply channel is diluted with a diluent supplied from the other channel to form self-assembled material particles, as described in any of 1 to 7. 9. A method for producing self-assembled material particles, comprising the step of diluting a self-assembled material-containing solution with a diluent to obtain a liquid containing self-assembled material particles, characterized in that the step is carried out using the continuous flow microfluidic system described in 8. 10. The method for producing the self-assembling material described in 9, wherein the self-assembling material is a lipid or an amphiphilic substance. 11. A method for producing the product according to 9 or 10, wherein the diluent is selected from aqueous solutions, buffer solutions, nucleic acid-containing aqueous solutions, protein-containing aqueous solutions, peptide-containing aqueous solutions, adjuvant-containing aqueous solutions, and mixtures thereof. [Effects of the Invention]

[0012] According to the continuous flow microfluidic system of the present invention, even when a fluid sample is fed into a microfluidic chip at a high pressure, self-assembled substance particles such as lipid nanoparticles with high size uniformity can be produced well and stably, and mass production of such self-assembled substance particles becomes possible.

Brief Description of the Drawings

[0013] [Figure 1] It is a schematic diagram showing an example of the continuous flow microfluidic system of the present invention. [Figure 2] It is a schematic diagram showing an example of the continuous flow microfluidic system of the present invention provided with a dilution section. [Figure 3] It is a perspective view showing an example of the microfluidic device constituting the continuous flow microfluidic system of the present invention. [Figure 4] It is an exploded perspective view showing the same microfluidic device. [Figure 5] It is a schematic diagram showing an example of the bent flow path section provided in the microfluidic chip constituting the continuous flow microfluidic system of the present invention.

Embodiments for Carrying Out the Invention

[0014] Hereinafter, the present invention will be described in more detail. The continuous flow microfluidic system of the present invention includes, for example, as shown in FIG. 1, a first continuous flow pump 11 that continuously supplies a first fluid, a second continuous flow pump 12 that continuously supplies a second fluid, and a microfluidic device 2 including a microfluidic chip 3.

[0015] The first continuous flow pump 11 has its suction side connected to the first reservoir 112 and its discharge side connected to the microfluidic device 2, and supplies the first fluid contained in the first reservoir 112 to the microfluidic device 2. The second continuous flow pump 12 has its suction side connected to the second reservoir 122 and its discharge side connected to the microfluidic device 2, and supplies the second fluid contained in the second reservoir 122 to the microfluidic device 2.

[0016] The first and second continuous flow pumps 11 and 12 are independent continuous flow pumps and are not particularly limited in type. For example, positive displacement pumps such as piston pumps, plunger pumps, diaphragm pumps, gear pumps, screw pumps, and vane pumps, or non-positive displacement pumps such as centrifugal pumps, turbine pumps, axial flow pumps, mixed flow pumps, and cascade pumps can be used.

[0017] As described above, the microfluidic device 2 includes a microfluidic chip 3. For example, as shown in Figures 3 and 4, an example of a device can be provided that includes a microfluidic chip 3 and a chip holder 21 that holds the microfluidic chip 3 internally.

[0018] The chip holder 21 has a structure in which a plate-shaped cover 211 and a base 212 are stacked and fixed with a plurality (10 in the figure) of fasteners (e.g., fixing screws) 22. Furthermore, the cover 211 and the base 212 may each be provided with recesses 213 and 214 on their opposing inner surfaces when stacked, for accommodating and holding the microfluidic chip 3. In addition, the recess 213 of the cover 211 is provided with screw holes 215, 216, and 217 that open on both the inner and outer surfaces, and short-shaft bolt-shaped connectors 23, 24, and 25 are screwed into these screw holes 215, 216, and 217, and the connectors 23, 24, and 25 have flow holes that open at both ends and are provided along the axis.

[0019] The cover 211 and base 212 described above typically have a plate-like shape. For ease of manufacturing, the inner surface shape of the cover 211 and base 212 that contact the microfluidic chip 3 is preferably a rectangular or other square shape, a circle, etc. The inner surface of the cover 211 and base 212 may be the same shape and size as the surface of the microfluidic chip 3 that contacts them, but it is preferable that it be the same shape as the surface of the microfluidic chip 3 and larger than the surface of the microfluidic chip 3. On the other hand, the thickness of the cover 211 and base 212 is not particularly limited, but is preferably 1 mm or more, more preferably 3 mm or more, even more preferably 5 mm or more, preferably 300 mm or less, more preferably 100 mm or less, and even more preferably 30 mm or less. A thickness within this range ensures the rigidity of the cover 211 and base 212, reduces damage during handling, and allows for weight reduction of the entire microfluidic device 2. Such a plate-like shape makes it easier to withstand the delivery of samples at high pressure and high flow rates.

[0020] As described above, recesses 213 and 214 may be formed on the respective surfaces of the cover 211 and base 212 facing the microfluidic chip 3. The microfluidic chip 3 can be fitted into these recesses 213 and 214, in which case the surface of the microfluidic chip 3 will be in contact with the recesses 213, and the surface of the microfluidic chip 3 will be in contact with the recesses 214. The depth of the recesses 213 and 214 is preferably 10% or more, more preferably 20% or more, preferably 50% or less, and more preferably 45% or less of the thickness of the microfluidic chip 3 (between one surface and the other). Furthermore, the size of the recesses 213 and 214 in the direction perpendicular to the depth direction is preferably 0.01 mm or more, more preferably 0.05 mm or more, preferably 0.5 mm or less, and more preferably 0.1 mm or less than the size of the surface of the microfluidic chip 3 that is in contact with the cover 211 and base 212. This makes it easier to align the microfluidic chip 3 and prevents misalignment of the microfluidic chip 3, thus maintaining good liquid-tightness, especially at the connector portion. Furthermore, it prevents damage to the microfluidic chip 3 due to excessive contact with the sides or circumferential surfaces of the recesses 213 and 214 of the chip holder 21.

[0021] The cover 211 and base 212 are preferably formed from a metallic material, a non-metallic material, or a composite material of a metallic and non-metallic material. Examples of metallic materials include chromium steel, stainless steel, aluminum, aluminum alloys, titanium, and titanium alloys; examples of non-metallic materials include ceramics; and examples of composite materials of a metallic and non-metallic material include fiber-reinforced metals and fiber-reinforced plastics. Among these, stainless steel is particularly preferred from the viewpoint of ease of processing, corrosion resistance, and heat resistance. Furthermore, the materials constituting the cover 211 and base 212 are preferably materials having a Young's modulus of 60 GPa or higher, and preferably 500 GPa or lower.

[0022] The fixing device 22 connects the cover 211 and the base 212, sandwiches the microfluidic chip 3 between the cover 211 and the base 212, and fixes the microfluidic chip 3 in close contact with the cover 211 and the base 212. The fixing method by the fixing device 22 is not particularly limited, as long as it can firmly fix the microfluidic chip 3 in close contact with the cover 211 and the base 212, but mechanical fixing with fixing screws is preferred, for example. When fixing with screws, through holes or non-through holes should be provided in the cover 211 and the base 212, and one or both of the cover 211 and the base 212 should be screw-shaped. With mechanical fixing with screws, the pressing force on the microfluidic chip 3 by the cover 211 and the base 212 can be adjusted for the entire microfluidic device 2 by adjusting the tightness of each screw.

[0023] Furthermore, the connectors 23, 24, and 25 preferably include a screw-shaped pressing member and a ring-shaped ferrule into which the tube is inserted. In such a connector, the ferrule can be configured to adhere tightly to the surface of the microfluidic chip 3 and the tube, respectively, by pressing from the pressing member. This configuration allows for good liquid-tightness between the surface of the microfluidic chip 3 and the tube.

[0024] The pressing members of connectors 23, 24, and 25 are preferably made of a resin material, but they can also be made of a metal material such as stainless steel. Examples of resin materials include PEEK, PPS, POM, PE, PP, ETFE, PCTFE, PTFE, and PFA.

[0025] The ferrule is preferably made of a resin material. Suitable resin materials include, for example, PEEK, PP, ETFE, and PCTFE. The material constituting the ferrule is preferably a material having a tensile strength of 20 MPa or more, more preferably 30 MPa or more, more preferably 300 MPa or less, and more preferably 200 MPa or less. If the tensile strength of the ferrule is within this range, the ferrule can be more reliably pressed against the surface of the microfluidic chip and the tube by the pressure from the pressing member, and good liquid tightness can be maintained.

[0026] A tube can be connected to the other end of the connector. The tube is preferably made of a resin material, but it can also be made of a metal material such as stainless steel. Examples of resin materials include PEEK, PTFE, and PFA.

[0027] The microfluidic chip 3 comprises a first supply channel 31 through which the first fluid flows and a second supply channel 32 through which the second fluid flows. The first and second supply channels 31 and 32 are joined at their downstream ends so that the two fluids merge, and a mixing and dilution channel 33 is provided downstream of this merging point 34 through which a mixed fluid of the first and second fluids flows. As shown in Figure 4, the upstream ends of the first and second supply channels 31 and 32 are provided with openings 311 and 321 that open to the top surface of the chip, respectively, and the downstream end of the mixing and dilution channel 33 is similarly provided with an opening 331 that opens to the top surface of the chip.

[0028] The microfluidic chip 3 is positioned and fixed within the recesses 213 and 214 of the chip holder 21, which is connected and fixed in a superimposed state between the cover 211 and the base 212 by the fixing device 22. In this state, the upper surface of the microfluidic chip 3 and the inner surface of the recess 213 of the cover 211 are in liquid-tight contact, and the tips of the connectors 23, 24, and 25, which are screwed into the screw holes 215, 216, and 217 of the cover 211, are in close contact with the openings 311, 321, and 331, respectively. The flow holes provided in these connectors 23, 24, and 25 are in communication with the first supply channel 31, the second supply channel 32, and the mixing and dilution channel 33 through the respective openings 311, 321, and 331. In the example shown in Figures 3 and 4, the openings 311, 321, and 331 for the flow channels are formed on one side of the microfluidic chip 3. However, if any of these openings are formed on the other side, and openings exist on both sides of the microfluidic chip 3, then screw holes corresponding to the screw holes 215, 216, and 217 should be provided in the cover 211 and base 212, respectively, according to each opening, and connectors corresponding to connectors 23, 24, and 25 should be attached.

[0029] Here, in the microfluidic device 2, although not particularly limited, the flatness of the surface in contact with the cover 211 of the microfluidic chip 3 and the flatness of the surface in contact with the base 212 of the microfluidic chip 3 are preferably 50 μm or less, more preferably 30 μm or less, even more preferably 20 μm or less, particularly preferably 15 μm or less, and most preferably 10 μm or less. Thickness variation (TTV: total thickness variation) can be applied to this flatness.

[0030] Furthermore, although not particularly limited, the flatness of the surface of the cover 211 in contact with the microfluidic chip 3 and the flatness of the surface of the base 212 in contact with the microfluidic chip 3 are preferably 50 μm or less, more preferably 30 μm or less, even more preferably 20 μm or less, particularly preferably 15 μm or less, and most preferably 10 μm or less. The flatness specified in JIS B 0621 can be applied to this flatness.

[0031] By adjusting these flatness and planarity as described above, even when a fluid sample is introduced into the microfluidic chip 3 at high pressure, the tensile and compressive stresses applied to the microfluidic chip 3 are effectively distributed to the chip holder 21, reducing the load on the microfluidic chip 3 itself. This makes the microfluidic chip 3 less prone to deformation, effectively suppressing liquid leakage at the contact points and damage to the microfluidic chip, allowing for the production of the target product more efficiently and stably.

[0032] Furthermore, although not particularly limited, the flatness of the portions of the microfluidic chip 3 surface where the tips of the connectors 23, 24, and 25 make contact is preferably 50 μm or less, more preferably 30 μm or less, even more preferably 20 μm or less, particularly preferably 15 μm or less, and most preferably 10 μm or less. Thickness variation (TTV) is applied to this flatness.

[0033] By adjusting the flatness of the surface of the microfluidic chip 3, which is in contact with the tips of connectors 23, 24, and 25, as described above, the microfluidic chip 3 becomes less susceptible to damage, and the liquid-tightness at the connection point between the microfluidic chip 3 and the flow path is increased, making it less likely for liquid leakage to occur even when a fluid sample is introduced into the flow path of the microfluidic chip at high pressure.

[0034] The flatness of the surface of the microfluidic chip 3, and the flatness of the surfaces of the cover 211 and base 212 can be adjusted by polishing the surfaces of the microfluidic chip 3, cover 211 and base 212. The flatness of the surface of the microfluidic chip 3, and the flatness of the surfaces of the cover 211 and base 212 only need to be such that they have the predetermined flatness or flatness at least in the parts where the microfluidic chip 3 and the cover 211 and base 212 are in contact, and furthermore, the flatness of the surface of the microfluidic chip 3 only needs to be such that they have the predetermined flatness in the parts where the tips of the connectors 23, 24, and 25 are in contact.

[0035] The above-mentioned microfluidic chip 3 is not particularly limited, but is preferably made of synthetic quartz glass from the viewpoint of long-term stability, weather resistance, and chemical resistance. Synthetic quartz glass can be obtained by taking a synthetic quartz glass ingot manufactured by a conventional method, shaping it to a predetermined size and thickness, and then, if necessary, applying lapping, rough polishing, or fine polishing to the surface.

[0036] Suitable cross-sectional shapes for the channels 31, 32, and 33 formed inside the microfluidic chip 3 include square, circular, semicircular, and approximately semicircular shapes. The length, width, and height of the channels can be appropriately selected according to the application of the microfluidic chip 3 used, but the width is usually 0.01 μm or more and usually 100,000 μm or less, and the height is usually 0.01 μm or more and usually 100,000 μm or less. The height is usually formed to be about 90% or less of the thickness of the microfluidic chip 3.

[0037] The openings 311, 321, and 331 of the microfluidic chip 3 described above only need to communicate with the flow channels 31, 32, and 33, and their size is not particularly limited. Suitable shapes for the openings include circular and angular shapes. The size of the openings (size along the surface of the microfluidic chip) is not particularly limited as described above, but from a manufacturing or handling standpoint, it is preferable that the length of one side of an angular opening is 0.1 to 5 mm, and the diameter of a circular opening is 0.1 to 5 mm.

[0038] The microfluidic chip 3 described above usually has a plate-like shape. The shape of the surface of the microfluidic chip 3 that contacts the cover 211 and base 212 is preferably a rectangular or other square shape, or a circular shape, for ease of manufacturing. With such a shape, good contact with the cover 211 and base 212 is ensured, and the microfluidic device can be reliably constructed. The size of the surface of the microfluidic chip 3 is not particularly limited, but for example, if it is square, the length of one side is preferably 10 to 1000 mm, and if it is circular, the diameter is preferably 10 to 1000 mm. On the other hand, the thickness of the microfluidic chip 3 is not particularly limited, but is preferably 0.01 mm or more, more preferably 0.1 mm or more, even more preferably 0.5 mm or more, preferably 300 mm or less, more preferably 100 mm or less, and even more preferably 15 mm or less. With a thickness in this range, the rigidity of the microfluidic chip 3 can be ensured, damage during handling can be reduced, and furthermore, the weight of the microfluidic chip 3 can be reduced.

[0039] By configuring the microfluidic device 2 in this way, fluid communication is improved between the discharge ports of the continuous flow pumps 11 and 12 and the supply port of the microfluidic device 2, as well as between the discharge port of the microfluidic device and the system outlet. This makes it possible to suppress pulsation in continuous flow even when the fluid sample is introduced at high pressure.

[0040] Furthermore, in the microfluidic chip 3 described above, the first fluid flowing through the first supply channel 31 and the second fluid flowing through the second supply channel 32 merge at the confluence point 34 and are mixed and diluted as they flow through the mixing and diluting channel 33. Here, the structure of this mixing and diluting channel 33 is not particularly limited, and for example, it may be a known three-dimensional micromixer structure capable of achieving instantaneous mixing of two liquids. However, when manufacturing self-assembled material particles, it is preferable to use a simple two-dimensional channel structure in which baffles (obstruction plates) of a constant width relative to the channel width are arranged alternately from both sides, as described in, for example, WO2018-190423, in order to form lipid nanoparticles with high size uniformity and particle size controllability.

[0041] Specifically, a flow channel structure having a bent flow channel section 6 as shown in Figure 5 can be preferably used. In other words, as described above, the flow channel of this microfluidic chip 3 consists of a first supply flow channel 31 that supplies a first fluid and a second supply flow channel 32 that supplies a second fluid, both independent of each other, which merge at a certain length on the upstream side (left side of the drawing). A single mixing and diluting flow channel 33 is formed downstream from this confluence point 34, and it is preferable that at least a part of this mixing and diluting flow channel 33 has a two-dimensionally bent flow channel section 6.

[0042] This bent channel section 6 can be composed of, for example, a plurality of structural elements 61 arranged to protrude inward from the side walls facing each other along the Y direction and spaced apart at predetermined intervals along the X direction, when the axial direction or extension direction of the mixing and diluting channel 33 upstream of the bent channel section 6 is defined as the X direction, and the width direction of the diluting channel perpendicular to the X direction is defined as the Y direction.

[0043] The structural elements 61 that form the bent channel section 6 preferably have a protruding width h in the Y direction within the mixed dilution channel, which is 1 / 2[y0] or more and less than 1[y0], when the width in the Y direction of the mixed dilution channel 33 upstream of the bent channel section 6 is [y0], more preferably 1 / 2[y0] or more and 39 / 40[y0] or less, and even more preferably 1 / 2[y0] or more and 3 / 4[y0] or less. As a result, channels y1 with a width greater than 0 and less than or equal to 1 / 2[y0] of the upstream channel width y0 are alternately formed at predetermined intervals. The protruding width h of the structural elements 61 does not need to be the same for all structural elements 61, and may be different as long as the predetermined conditions described above are met. The channel width y1 formed in this way may also be different at each location where the structural elements 61 are formed. For example, as you go downstream, the protruding width h of each structural element 61 may gradually increase, and the channel width y1 may gradually narrow. In the areas where each of these structural elements 61 is present, the flow path becomes bent, and the flow path width y1 is narrowed to a width of 1 / 2[y0] or less, thereby improving the mixing efficiency and the efficiency of molecular diffusion in the fluid.

[0044] Furthermore, the width w in the X direction of the structural element 61 may differ for each structural element 61 or may be a constant width, and the distance d between each structural element 61 may also differ or be at a constant interval. For example, the width w and distance d may gradually increase or decrease as one moves downstream. The width w in the X direction of the structural element 61 is preferably about 1 / 10[y0] or more and 5[y0] or less, when the width [y0] in the Y direction of the mixing and diluting channel 33 upstream of the bent channel section 6 is defined as [y0]. In addition, the distance x0 from the confluence point 34 of the first and second supply channels 31 and 32 to the first structural element 61 is not particularly restricted and can be set as appropriate, but it is preferable to set a distance equal to or greater than the width w of the structural element.

[0045] The specific values ​​of the protruding width h, width w in the X direction, and distance d between each structural element of the above-mentioned structural element 61 are not particularly limited, and depend on factors such as the size of the particles, the number of structural elements 61, the length and width of the mixing and dilution channel 33, and other conditions, especially when trying to obtain self-assembled material particles such as lipid particles. Specifically, they are as follows:

[0046] For example, if the channel width [y0] of the upstream mixing and dilution channel 33 is 200 μm, it is preferable that the protruding width h of the structural element 6 be 100 or more and less than 200 μm. Therefore, in the bent section 6 where each structural element 61 exists, it is preferable that the channel width y1 is greater than 0 and about 100 μm or less. Also, if the channel width [y0] on the upstream side of the mixing and dilution channel 33 is 20 to 1000 μm, typically 200 μm, it is preferable that the width w of each structural element 61 be about 20 to 1000 μm. In this case, the width w of each structural element 61 does not necessarily have to be the same, and may be different as described above.

[0047] Furthermore, the spacing d between each structural element 61 is influenced by the size of the lipid particles to be obtained, the number of structural elements 61, their height h, width w, and other conditions, but it is preferable that the spacing d between each structural element 61 is between 1 / 10[y0] and 5[y0] of the upstream channel width[y0] of the mixing and dilution channel 33. Specifically, for example, if the upstream channel width[y0] is 20 to 1000 μm, typically 200 μm, it is desirable that the spacing d between adjacent structural elements 61 be approximately 20 to 1000 μm. The spacing d between adjacent structural elements 61 does not necessarily have to be the same; they may be different. For example, the spacing d may gradually narrow as you move downstream.

[0048] The channel width y0 of the mixing and diluting channel 33 from the confluence point 34 of the first supply channel 31 and the second supply channel 32 to the first structural element 61 is not particularly limited, but is preferably about 0.01 to 100,000 μm, more preferably about 10 to 10,000 μm, from the viewpoint of mass production of lipid nanoparticles with high size uniformity in the production of self-assembled material particles, for example.

[0049] In Figure 5, the structural elements 61 constituting the bent channel section 6 have walls perpendicular to the X direction along the axis of the mixing and diluting channel 33. However, this angle does not necessarily have to be exactly 90°, and a somewhat inclined angle can also be an effective configuration. There are no particular limitations, but specifically, for example, an angle of about 30 to 150° is preferable, more preferably 40 to 140°, and even more preferably 80 to 100° is well acceptable. Furthermore, the shape of the corners on the channel side of each structural element 61 can also be somewhat rounded, and there are no particular limitations, but for example, an R of 50 μm or less, and more preferably R of 20 μm or less, may be acceptable. However, in order to obtain nano-sized lipid particles with higher controllability and uniformity, it is desirable to keep these tolerance values ​​as small as possible. Furthermore, in the embodiment shown in Figure 5, the X direction, which is the axial direction or extension direction of the mixing and diluting channel 33 in this channel structure, is conveniently represented as a straight line. However, this X direction merely indicates the axial direction of the mixing and diluting channel 33, and in reality, it is not limited to such a straight line; for example, it may be curved with a certain curvature. The Y direction, which is the width direction of the mixing and diluting channel 33 that is perpendicular to this curved X direction, refers to the direction perpendicular to the tangent to the axis of that part.

[0050] Furthermore, in the example shown in Figure 5, the bent portion 6 is formed by arranging approximately rectangular baffles (structural elements 61) alternately from both sides of the flow path. However, it is not limited to being constructed by arranging separate baffles on the flow path in this manner. That is, as long as a flow path of a similar shape is formed to correspond to the flow path formed by arranging such baffles, the configuration of the structural elements 61 is not particularly limited. It may also be formed by integrally forming the wall surface of the mixing and diluting flow path 33 (while maintaining a nearly constant wall thickness) while bending it into a predetermined shape to constitute the structural elements 61 described above, thereby creating a two-dimensional flow path shape that bends and contracts according to the aforementioned provisions. Naturally, the bent flow path portion 6 in the present invention includes such configurations.

[0051] Furthermore, since the flow channel structure composed of the first and second supply channels 31 and 32 and the mixing and dilution channel 33 is a two-dimensional flow channel structure as described above, the dimensions of the flow channel in the depth direction (paper thickness direction in Figure 5) are not particularly limited, but are preferably about 0.01 to 100,000 μm, more preferably about 10 to 1,000 μm.

[0052] In Figures 3-5, a flow channel structure consisting of two supply channels 31 and 32 and one mixing / diluting channel 33 is shown. However, in the present invention, it is sufficient for multiple supply channels, each having a certain length, to merge and form one mixing / diluting channel. For example, there may be three supply channels. When there are three supply channels, it is preferable that the first supply channel 31, the second supply channel 32, and the third supply channel each have a certain length and merge to form one mixing / diluting channel 33, so that the first fluid introduced from the first supply channel 31 comes into contact with the third fluid introduced from the third supply channel before it merges with the second fluid introduced from the second supply channel 32.

[0053] The mixing and dilution of fluids in this channel structure depends on molecular diffusion, for example, when producing self-assembled material particles such as lipid particles. The faster the dilution rate of the raw material lipid solution, the smaller the size of the resulting lipid particles. Therefore, by adjusting the protrusion width h, width w, and arrangement of each of the above-mentioned structural elements 61 (baffles), the dilution rate of the raw material solution can be controlled, making it possible to form nanoparticles with higher particle size controllability than conventional methods.

[0054] Up to this point, the microfluidic device 2 has been described with reference to Figures 3 to 5. However, the microfluidic device in the present invention is not limited to the microfluidic device 2 shown in Figures 3 to 5. For example, the shape and material of the cover 211 and base 212 that constitute the chip holder 21, the shape and material of the microfluidic chip 3 and the configuration of the flow path, and the method and form of fixing the microfluidic chip 3 to the chip holder 21 may be changed as appropriate, as long as they do not depart from the spirit of the present invention.

[0055] The discharge side of the first continuous flow pump 11 is connected to the connector 23 of the microfluidic device 2, and as shown in Figure 1, the first continuous flow pump 11 supplies the first fluid from the first reservoir 112 to the first supply channel 31 of the microfluidic chip 2 via the connector 23. The discharge side of the second continuous flow pump 12 is connected to the connector 24 of the microfluidic device 2, and as shown in Figure 1, the second continuous flow pump 12 supplies the second fluid from the second reservoir 122 to the second supply channel 32 of the microfluidic chip 2 via the connector 24.

[0056] The first fluid introduced into the first supply channel 31 and the second fluid introduced into the second supply channel 32 merge at the confluence point 34 of the channels and are mixed as they flow through the mixing and diluting channel 33. The mixed fluid is then discharged from the microfluidic device 2 through the connector 25 at the downstream end of the mixing and diluting channel 33. A discharge channel 4 is connected to the connector 25 at the downstream end of the mixing and diluting channel 33, and the mixed fluid discharged from the microfluidic device 2 is collected in the sample container 51 through this discharge channel 4.

[0057] Furthermore, as shown in Figure 1, a waste channel 523 can be provided in the discharge channel 4, for example, via a waste valve 521. This allows waste to be collected into the waste container 52 through the waste channel 523 during system priming or other non-productive operations (for example, operation until particle production stabilizes or operation for cleaning the channel).

[0058] Furthermore, although not particularly limited, the system of the present invention may include a dilution section 7 for introducing a dilution solution in the middle of the discharge channel 4 (upstream of the waste valve if the waste channel 523 is provided), as shown in Figure 2. This dilution section 7 is equipped with a third continuous flow pump 71 that continuously supplies the dilution solution, and its suction side is connected to a third reservoir 72 that contains the dilution solution, while its discharge side is connected to the discharge channel 4 via a dilution channel 74 and a T-connector 73.

[0059] By providing this dilution unit 7, the dilution solution can be continuously added to the mixed fluid flowing through the discharge channel 4, for example, to adjust the pH of the solution, thereby stabilizing the manufactured particles. In the system shown in Figure 2, the components other than the dilution unit 7 are the same as those in the system in Figure 1, and the same reference numerals are used for the same parts, and their explanations are omitted.

[0060] The continuous flow microfluidic system of the present invention can be applied to various applications as long as it involves mixing a first fluid and a second fluid while allowing a small amount of fluid to flow. For example, it is particularly suitable for applications where a solution containing self-assembling material particles such as lipids or amphiphilic substances is continuously introduced as the first fluid, and a diluent is continuously introduced as the second fluid, thereby diluting the self-assembling material-containing solution in the first fluid with the diluent in the second fluid to form the desired self-assembling material particles.

[0061] In this system, the pulsation rate of the continuous flow formed by the system is set to 5% or less, preferably 1% or less, and more preferably 0.5% or less. The pulsation rate (Xs) of the continuous flow formed by the system is calculated by the following formula (1).

[0062]

number

[0063] Furthermore, it is preferable to check the pulsation rate of the continuous flow formed by the present invention system between the discharge ports of the continuous flow pumps 11 and 12 and the supply section to the microfluidic device 2 (connectors 23 and 24 in the example shown in Figures 1 to 4), and between the discharge section from the microfluidic device 2 (connector 25 in the example shown in Figures 1 to 4) and the system outlet (downstream end of the discharge channel 4 in the example shown in Figures 1 to 4).

[0064] If the pulsation rate of this continuous flow exceeds 5%, mixing proceeds slowly before and after the confluence point 34 (see Figures 4 and 5), and especially between the confluence point 34 and the bent channel section 6 of the mixing and diluting channel 33 if the bent channel section 6 is provided. This makes it easier for coarse particles to form, reducing the size uniformity of lipid nanoparticles. There is no particular lower limit to this pulsation rate, but a lower rate is preferable.

[0065] To control the pulsation rate of the continuous flow formed by the system of the present invention described above, the pulsation rate of the continuous flow pump is preferably 5% or less, more preferably 1% or less, and even more preferably 0.5% or less. The pulsation rate (Xp) of the continuous flow pump is calculated by the following formula (2).

number

[0066] If the pulsation rate of the continuous flow pump is less than or equal to the value mentioned above, gradual mixing can be suppressed between the microfluidic device 2 and the mixing / dilution channel 33.

[0067] The system of the present invention may utilize a software system for controlling manufacturing parameters. These manufacturing parameters may include, but are not limited to, fluid flow rate, the ratio of independent fluid flow rates, pressure within the apparatus, and temperature control. Such software controls are generally known to those skilled in the art.

[0068] Next, the method for producing self-assembled material particles of the present invention will be described. The production method of the present invention includes the step of diluting a self-assembled material-containing solution with a diluent to obtain a liquid containing self-assembled material particles, and this step is carried out using the continuous flow microfluidic system of the present invention described above.

[0069] Specifically, referring to Figures 1-5, the first continuous flow pump 11 supplies the self-assembling material-containing solution from the first reservoir 112 to the microfluidic device 2, and the second continuous flow pump 12 supplies the diluent from the second reservoir 122 to the microfluidic device 2. The self-assembling material-containing solution supplied from the first continuous flow pump 11 flows into the first supply channel 31 of the microfluidic chip 3 via the connector 23 of the microfluidic device 2, and the diluent supplied from the second continuous flow pump 12 flows into the second supply channel 32 of the microfluidic chip 3 via the connector 24 of the microfluidic device 2. The self-assembling material-containing solution flowing through the first supply channel 31 and the diluent flowing through the second supply channel 32 merge and come into contact at the confluence point 34 of the two supply channels 23 and 24, becoming a mixed fluid and flowing through the mixed diluent channel 33. At this time, the self-assembling material-containing solution is diluted by the diluent to form self-assembling material particles. The mixed fluid containing these formed self-assembled material particles is then discharged from the microfluidic device 2 via the connector 25 at the downstream end of the mixing and dilution channel 33 and collected in the sample container 51 through the discharge channel 4.

[0070] The self-assembled material particles formed in the present invention are particles that contain the self-assembled material as a particle component. Particles that contain the self-assembled material as a particle component are obtained by the association of self-assembled materials with each other to form particles, and the encapsulated material that coexists in the system during particle formation can also be incorporated into the particles. The constituent components of particles formed under conditions in which the encapsulated material coexists are at least the self-assembled material and the encapsulated material.

[0071] The self-assembling material-containing solution and diluent can be introduced into a single mixing and diluting channel 33, for example, so that the total flow rate is 3 L / h or more. However, the total flow rate is not limited to this range and can be appropriately determined considering the structure and dimensions of the channel structure, the type of self-assembling material-containing solution and diluent, the particle size of the desired self-assembling material particles, the encapsulation efficiency of the encapsulated material, etc. From the viewpoint of mass production of self-assembling material particles with high size uniformity, the total flow rate of the self-assembling material-containing solution and diluent can be in the range of 3 L / h to 20 L / h, for example.

[0072] The ratio (V1:V2) of the flow rate V1 of the self-assembling material-containing solution supplied as the first fluid to the flow rate V2 of the diluent medium supplied as the second fluid can be, for example, in the range of 1:1 to 1:20. However, it is not limited to this range and can be appropriately selected within the range in which the desired particles can be obtained.

[0073] The self-assembling material-containing solution may be, for example, any solution selected from the group consisting of a neutral lipid-containing solution, anionic lipid-containing solution, cationic lipid-containing solution, and polymer-containing solution, but is not limited to these. The self-assembling material in the present invention may be any material that has a self-assembly function and, as described above, can associate with other self-assembling materials and form particles.

[0074] Examples of lipids that are self-assembling substances include, but are not limited to, naturally derived lipids such as soy lecithin, hydrogenated soy lecithin, egg yolk lecithin, phosphatidylcholines (e.g., egg-derived eggPC), phosphatidylserines, phosphatidylethanolamines, phosphatidylinositols, phosphasphingomyelins, phosphatidic acids, long-chain alkyl phosphates, gangliosides, glycolipids, phosphatidylglycerols, sphingolipids, sterols, lysophospholipids, and non-naturally derived lipids. In addition, cationic non-natural lipids considered suitable as components of nucleic acid delivery liposomes include N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), 3-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), and N-(1,2-dimyristiloxypropane-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), and lipofectin (registered (Trademark), Lipofectamine (Registered Trademark), Transfectam (Registered Trademark), 1,2-Dioleoyl-3-dimethylammonium-propane (DODAP), 1,2-Dioleyloxy-3-dimethylaminopropane (DODMA), (N,N-dimethyl-2,3-bis(tetradecyloxy)propane-1-amine (DMDMA), 1,2-Dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-Dilinoleyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-Dilinoleyloxy-3-morpholinopropane (DLin-MA), 1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 2,2-Dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-Dioxolane (DLin-KC2-DMA), (6Z,9Z,28Z,31Z)-Heptatriaconta-6,9,28,31-Tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA), 1,2-Dilinoleyloxy-3-trimethylaminopropane chloride (DLin-TMA-Cl), 1,2-Dilinoleoyl-3-trimethylaminopropane chloride (DLin-TAP-Cl), 1 ,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanediol (dio) (DOAP), 1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), and 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), etc. can be used. The above-mentioned 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA) and their analogues are described in Japanese Patent Publication No. 2013-245190, Japanese Patent Publication No. 2016-84297, and Japanese Patent Publication No. 2019-151589.

[0075] Furthermore, the self-assembling material may also be an amphiphilic material. Examples of amphiphilic materials that are self-assembling materials are not particularly limited, but examples include amphiphilic polymer compounds, such as amphiphilic block copolymers like polystyrene-polyethylene oxide block copolymer, polyethylene oxide-polypropylene oxide block copolymer, polylactic acid polyethylene glycol copolymer, and polycaprolactone-polyethylene glycol copolymer.

[0076] The above-mentioned encapsulated substances are not particularly limited, but include biomacromolecules such as nucleic acids, peptides, proteins, and glycans, metal ions, low-molecular-weight or medium-molecular-weight organic compounds, organometallic complexes, and metal particles. From the perspective of application, examples include drugs such as anticancer agents, antioxidants, antibacterial agents, anti-inflammatory agents, vitamins, artificial blood (hemoglobin), vaccines, hair growth agents, moisturizers, dyes, whitening agents, and pigments, as well as physiologically active substances and cosmetics. If these encapsulated substances are water-soluble, they can be contained within the aqueous phase of the formed particles. In the case of water-soluble charged substances, aggregates can be formed with self-assembling substances having the opposite charge, and at the same time, these aggregates can be used as a core to form self-assembling substance particles which can then be contained within the particles. If the substance is poorly soluble in water, it can be contained within the hydrophobic portion of the self-assembling membrane formed by the self-assembling substance, or it can be contained within the particles as an aggregate formed by bonding with the hydrophobic portion of the self-assembling substance.

[0077] The water-miscible organic solvent used to dissolve the self-assembling material and prepare a particle solution is not particularly limited, but examples include water-miscible organic solvents such as alcohols, ethers, esters, ketones, and acetals. In particular, alcohols such as methanol, ethanol, t-butanol, butanediols, 1-propanol, 2-propanol, and 2-butoxyethanol, especially alkanols having 1 to 6 carbon atoms, are preferred. Other examples include ethers such as tetrahydrofuran, acetonitrile, and acetone.

[0078] Next, as the diluent medium used as the second fluid, water, or aqueous solutions that are basically mainly composed of water, such as physiological saline, phosphate buffer solution, acetate buffer solution, citrate buffer solution, malate buffer solution, 2-morpholinoethanesulfonic acid (MES), 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), trishydroxymethylaminomethane (Tris), ethylenediaminetetraacetic acid (EDTA), etc., are used as appropriate depending on the intended use of the particles to be formed. In addition to aqueous solutions and buffer solutions, the diluent medium may also be a solution containing water-soluble substances. Examples of water-soluble substances include low molecular weight substances, medium molecular weight substances, high molecular weight substances, nucleic acids, proteins, peptides, and physiologically active substances containing them, pharmaceuticals, cosmetic materials, vaccines, adjuvants, etc. Examples of diluents further containing water-soluble substances include aqueous solutions containing low molecular weight substances, aqueous solutions containing medium molecular weight substances, aqueous solutions containing high molecular weight substances, aqueous solutions containing nucleic acids, aqueous solutions containing proteins, aqueous solutions containing peptides, aqueous solutions containing physiologically active substances, aqueous solutions containing pharmaceuticals, aqueous solutions containing cosmetic materials, aqueous solutions containing vaccines, aqueous solutions containing adjuvants, and mixtures thereof. Among these, aqueous solutions, buffer solutions, aqueous solutions containing nucleic acids, aqueous solutions containing proteins, aqueous solutions containing peptides, and aqueous solutions containing adjuvants are particularly preferred.

[0079] The self-assembled material particles obtained by the method of the present invention can be nano-sized, and particles with a Z-mean particle diameter in the range of, for example, 10 to 1000 nm can be obtained, and furthermore, particles in the range of 20 to 200 nm can be obtained. However, it is not limited to this range. The Z-mean particle diameter is also called the cumulative average (harmonic intensity averaged over the particle diameters) and is defined in ISO 13321. [Examples]

[0080] The present invention will be described in more detail below with reference to examples and comparative examples, but the present invention is not limited to the following examples.

[0081] [Example 1, Comparative Example 1, Reference Examples 1, 2] Lipid nanoparticles were produced using the continuous flow microfluidic system shown in Figure 1, as described below.

[0082] <Continuous Flow Microfluidic System> The continuous flow microfluidic system, as shown in Figure 1, includes two continuous flow pumps 11 and 12, a microfluidic device 2, a discharge channel 4, a waste valve 521, and a waste channel 523, with tubes connecting both reservoirs 112 and 122 to the outlet of discharge channel 4. The pulsation rate of the continuous flow formed by this system was adjusted as shown in Table 1.

[0083] The microfluidic device 2 comprising this system is as shown in Figures 3 and 4, and includes a microfluidic chip 3, a chip holder 21 (cover 211, base 212, and fixing device 22), and connectors 23, 24, and 25, as follows.

[0084] The microfluidic chip 3 is made of synthetic quartz glass measuring 30 mm x 70 mm and 1.8 mm thick. The flow channel is a flow channel structure with the basic structure shown in Figure 5, with a flow channel depth of 500 μm, a flow channel width y0 of 2000 μm, a height h of each structural element 61 of 1000 μm, a width w of each structural element 61 of 1000 μm, a spacing d between adjacent structural elements 61 of 1000 μm, and a total number of structural elements 61 of 20 (total length 60 mm). The supply holes 311, 321 and the discharge hole 331 (see Figure 4) are all circular in shape with a diameter of 2.0 mm.

[0085] The cover 211 and base 212 are each made of stainless steel (SUS304) measuring 60 mm x 100 mm and 7 mm thick. The recesses 213 and 214 into which the microfluidic chip 3 is fitted are both 30.1 mm x 70.1 mm and 0.5 mm deep. The screw-type fasteners 22 are M5 screws, and unlike in Figures 3 and 4, there are seven of them. Seven screw-shaped holes corresponding to the fasteners 22 are formed in each of the cover 211 and base 212. In addition, three screw-type (M6 screw) connectors 23, 24, and 25 are used, and three screw holes 215, 216, and 217 corresponding to the connectors are formed in the cover 211. The connector pressing members are made of PEEK, and the ferrules are made of PTFE. The tubes are also made of PEEK.

[0086] The flatness of the surface of the microfluidic chip 3 facing the cover 211 and base 212, the flatness of the surface of the cover 211 and base 212 facing the microfluidic chip 3, and the flatness of the surface of the microfluidic chip 3 at the point where the tips (ferrules) of connectors 23, 24, and 25 make contact were adjusted as shown in Table 1.

[0087] As shown in Figures 3 and 4, the microfluidic chip 3 was fitted into the recesses 213 and 214 of the cover 211 and base 212, respectively. The fastener 22 was screwed into the hole to connect the cover 211 and base 212, and the microfluidic chip 3 was secured by being sandwiched between the cover 211 and base 212. In addition, the connectors 23, 24, and 25 were screwed into the screw holes 215, 216, and 217 of the cover 211, respectively, and the ferrules of each connector were pressed to secure each connector 23, 24, and 25 to the cover 211 and bring them into contact with the surface of the microfluidic chip 3.

[0088] [Table 1]

[0089] <Manufacturing of lipid nanoparticles> The self-assembling material-containing solution described below was supplied to the system as the first fluid from the first reservoir 112 via the first continuous flow pump 11, and the diluent described below was supplied to the system as the second fluid from the second reservoir 122 via the second continuous flow pump 11, and lipid nanoparticles were produced under the following conditions.

[0090] • Self-assembling substance-containing solution: 10 mg / mL phosphatidylcholine solution in ethanol • Dilution medium: physiological saline Total flow rate: 120 mL / min ·Flow rate ratio: 1:3 (solution containing self-organizing substance: dilution medium) • Dialysis: D-PBS(-) ·Particle size measurement: DLS

[0091] In Example 1, no leakage or damage to the microfluidic chip was observed, and the production of highly uniform 20 nm lipid nanoparticles was confirmed. In Comparative Example 1, no leakage or damage to the microfluidic chip was observed, but coarse particles other than the target 20 nm particles were observed. In Reference Example 1, leakage was observed at the contact point between the microfluidic chip and the connector, which may have hindered the production of lipid nanoparticles. Furthermore, in Reference Example 2, damage occurred to the microfluidic chip under the above conditions, making it difficult to produce lipid nanoparticles. [Explanation of Symbols]

[0092] 11. First continuous flow pump 112 First Reservoir 12. Second continuous flow pump 122 Second Reservoir 2 Microfluidic Devices 21 Chip holder 211 Cover 212 base 213,214 recess 22 Fixtures 23, 24, 25 Connectors 3 Microfluidic Chips 31. First supply channel 311 Opening (supply hole) 32 Second supply channel 321 Opening (supply hole) 33 Mixing and Dilution Channel 331 Opening (exhaust hole) 34 Confluence 4. Discharge channel 51 Sample containers 52 waste containers 521 Waste Valve 523 Waste collection route 6. Bent channel section 61 Structure 7 Dilution portion 71. Third continuous flow pump 72 Third Reservoir 73 T-connector 74 Dilution channel

Claims

1. A continuous flow microfluidic system for continuous flow operation of a microfluidic chip, comprising the following (1) to (4), characterized in that the pulsation rate of the continuous flow formed by the system is 5% or less. (1) A first continuous flow pump that continuously supplies a first fluid from a first reservoir. (2) A second continuous flow pump that continuously supplies the second fluid from the second reservoir. (3) A microfluidic device comprising a microfluidic chip having the following channels (a) to (c). (a) A first supply channel through which the first fluid flows. (b) A second supply channel through which the second fluid flows. (c) A mixing and diluting channel provided downstream of the confluence point where the first supply channel and the second supply channel merge over a certain length, through which a mixed fluid obtained by mixing the first fluid and the second fluid flows. (4) A discharge channel through which the mixed fluid discharged from the microfluidic device flows.

2. The continuous flow microfluidic system according to claim 1, comprising a dilution section including a third continuous flow pump that continuously supplies a dilution solution from a third reservoir, and a dilution channel that supplies the dilution solution discharged from the third continuous flow pump downstream of the microfluidic device in the system to mix the dilution solution with the mixed fluid.

3. The continuous flow microfluidic system according to claim 1 or 2, further comprising a waste recovery channel branched from the discharge channel via a waste valve.

4. The continuous flow microfluidic system according to claim 1 or 2, having a processing capacity of at least 3 L / h per mixing and dilution channel.

5. The continuous flow microfluidic system according to claim 1 or 2, wherein at least a portion of the mixing and diluting channel has a bent channel section formed by a plurality of structural elements installed in the channel, and when the axial direction of the mixing and diluting channel is the X direction, the width direction of the mixing and diluting channel perpendicular to the X direction is the Y direction, and the width of the mixing and diluting channel upstream of the bent channel section in the Y direction is [y0], the structural elements, which are arranged to protrude inward from the side walls facing each other along the Y direction in the mixing and diluting channel, are installed alternately at predetermined intervals along the X direction, and the protrusion width of the structural elements in the Y direction is 1 / 2 [y0] or more and less than 1 [y0].

6. The continuous flow microfluidic system according to claim 1 or 2, wherein the microfluidic chip is made of synthetic quartz glass.

7. The microfluidic device comprises a chip holder and a connector. The chip holder comprises a cover and a base that contact the surface of the microfluidic chip, and a fastener that connects and fixes the cover and the base, the microfluidic chip is placed between the cover and the base and the cover and the base are connected and fixed by the fastener, and the microfluidic chip is fixed in close contact with the cover and the base, and the connector penetrates at least one of the cover and the base, with one end connected to an opening provided at the upstream end of the first supply channel, the upstream end of the second supply channel, and the downstream end of the mixing and diluting channel of the microfluidic chip, and the other end forming a supply port for the first fluid or the second fluid or an outlet for the mixed fluid, in a microfluidic device, The continuous flow microfluidic system according to claim 1 or 2, wherein the flatness of the surface of the microfluidic chip in contact with the cover and the flatness of the surface of the microfluidic chip in contact with the base are both 50 μm or less, and the flatness of the surface of the cover in contact with the microfluidic chip and the flatness of the surface of the base in contact with the microfluidic chip are both 50 μm or less.

8. A continuous flow microfluidic system for producing self-assembled material particles, wherein in the mixing and diluting channel, a self-assembled material-containing solution supplied from one of the first supply channel and the second supply channel is diluted with a diluent supplied from the other channel to form self-assembled material particles.

9. A method for producing self-assembled material particles, comprising the step of diluting a self-assembled material-containing solution with a diluent to obtain a liquid containing self-assembled material particles, wherein the step is carried out using the continuous flow microfluidic system described in claim 8.

10. The manufacturing method according to claim 9, wherein the self-assembling material is a lipid or an amphiphilic substance.

11. The manufacturing method according to claim 9, wherein the dilution medium is selected from aqueous solutions, buffer solutions, nucleic acid-containing aqueous solutions, protein-containing aqueous solutions, peptide-containing aqueous solutions, adjuvant-containing aqueous solutions, and mixtures thereof.

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